title: Simple, fast, and efficient method of manual oocyte enucleation using a pulled Pasteur pipette.
abstract: Cloning mammals by somatic cell nuclear transfer entails the replacement of oocyte chromosomes with the nucleus of a somatic cell. A major step in this technique is to efficiently produce large batches of enucleated oocytes, a process that requires considerable micromanipulation skills and expensive equipments. Here, a simple, fast, and efficient method of manual oocyte enucleation was introduced that can be adopted in every laboratory with the minimum equipments. Common laboratory glass pipettes were pulled on the flame of a burner and then used for manual bisection or enucleation of sheep and goat zona-free oocytes by passing them through the discontinuous cutting border of culture medium and mineral oil. The described techniques showed a certain efficiency to conveniently bisect or enucleate large batches of sheep, and goat oocytes being pre-treated with demecolcine. The method may be straightforward for simple manipulation of oocytes of other species and for development of automated cloning methods as well.
authors.affiliation: Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran, smhosseini@royaninstitute.org.
authors.forename: S M
authors.initials: SM
authors.lastname: Hosseini
authors.forename: F
authors.initials: F
authors.lastname: Moulavi
authors.forename: V
authors.initials: V
authors.lastname: Asgari
authors.forename: A
authors.initials: A
authors.lastname: Shirazi
authors.forename: A H
authors.initials: AH
authors.lastname: Abazari-Kia
authors.forename: H R
authors.initials: HR
authors.lastname: Ghanaei
authors.forename: M H
authors.initials: MH
authors.lastname: Nasr-Esfahani
mesh_terms: D000818:Animals; D002467:Cell Nucleus; D019976:Cloning, Organism; D005260:Female; D006041:Goats; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology*; D012756:Sheep